ABSTRACT Little is known about how novel multicellular developmental programs arise in evolution, largely because multicellularity arose deep in the past and early steps have been lost to extinction. The PI has circumvented this constraint by creating a new model system, using directed evolution to directly study the origin of multicellularity and development. The proposed research will help resolve two major knowledge gaps in the field of developmental biology: 1) How does development evolve de novo? 2) What are the long-term evolutionary consequences of development? After thousands of generations of directed evolution, the PI has observed the evolution of autonomous cell type specification in snowflake yeast, his model system of early multicellularity. Specifically, snowflake yeast form multicellular groups that are far more mechanically tough by adaptively differentiating into two cell types, in which cells deep in the cluster interior change their budding angle by 90 degrees and interlock neighboring cellular branches. In this model system (and in most simple multicellular organisms), a cell?s age provides key information about its location, allowing temporal changes in gene expression to drive spatial patterns of cellular differentiation. Preliminary data suggests that the chaperone protein Hsp90 has been co-opted to act as an age (and thus location)-dependent developmental switch, regulating the transition between cell types. The proposed research will further examine the evolution of this novel developmental mechanism, and will contextualize these wet-lab experiments with both 3D biophysical simulations and evolutionary models, allowing the PI to derive general principles from these experimental results. The proposed research will also examine how stochastic cellular behaviors can be co-opted for symmetry breaking, allowing snowflake yeast to evolve more complex multicellular structures. Finally, the proposed research will examine the evolutionary consequences of development: namely, how cellular differentiation can entrench a lineage into a multicellular state. This work will examine how cellular differentiation strips cells of their evolutionary autonomy by limiting the potential for reversion to unicellularity. Development would thus have a doubly-profound impact on an organism?s evolutionary dynamics: opening new avenues for increased multicellular complexity while closing off opportunities for ancestral, unicellular behaviors.